An Anti-Human ICAM-1 Antibody Inhibits Rhinovirus-Induced Exacerbations of Lung Inflammation

Human rhinoviruses (HRV) cause the majority of common colds and acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Effective therapies are urgently needed, but no licensed treatments or vaccines currently exist. Of the 100 identified serotypes, ∼90% bind domain 1 of human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, making this an attractive target for development of therapies; however, ICAM-1 domain 1 is also required for host defence and regulation of cell trafficking, principally via its major ligand LFA-1. Using a mouse anti-human ICAM-1 antibody (14C11) that specifically binds domain 1 of human ICAM-1, we show that 14C11 administered topically or systemically prevented entry of two major groups of rhinoviruses, HRV16 and HRV14, and reduced cellular inflammation, pro-inflammatory cytokine induction and virus load in vivo. 14C11 also reduced cellular inflammation and Th2 cytokine/chemokine production in a model of major group HRV-induced asthma exacerbation. Interestingly, 14C11 did not prevent cell adhesion via human ICAM-1/LFA-1 interactions in vitro, suggesting the epitope targeted by 14C11 was specific for viral entry. Thus a human ICAM-1 domain-1-specific antibody can prevent major group HRV entry and induction of airway inflammation in vivo

Discovery and characterisation of an antibody that selectively modulates the inhibitory activity of plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that regulates fibrinolysis, cell adhesion and cell motility via its interactions with plasminogen activators and vitronectin. PAI-1 has been shown to play a role in a number of diverse pathologies including cardiovascular diseases, obesity and cancer and is therefore an attractive therapeutic target. However the multiple patho-physiological roles of PAI-1, and understanding the relative contributions of these in any one disease setting, make the development of therapeutically relevant molecules challenging. Here we describe the identification and characterisation of fully human antibody MEDI-579, which binds with high affinity and specificity to the active form of human PAI-1. MEDI-579 specifically inhibits serine protease interactions with PAI-1 while conserving vitronectin binding. Crystallographic analysis reveals that this specificity is achieved through direct binding of MEDI-579 Fab to the reactive centre loop (RCL) of PAI-1 and at the same exosite used by both tissue and urokinase plasminogen activators (tPA and uPA). We propose that MEDI-579 acts by directly competing with proteases for RCL binding and as such is able to modulate the interaction of PAI-1 with tPA and uPA in a way not previously described for a human PAI-1 inhibitor

A synthetic lethal screen identifies a role for the cortical actin patch/endocytosis complex in the response to nutrient deprivation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae whi2Delta cells are unable to halt cell division in response to nutrient limitation and are sensitive to a wide variety of stresses. A synthetic lethal screen resulted in the isolation of siw mutants that had a phenotype similar to that of whi2Delta. Among these were mutations affecting SIW14, FEN2, SLT2, and THR4. Fluid-phase endocytosis is severely reduced or abolished in whi2Delta, siw14Delta, fen2Delta, and thr4Delta mutants. Furthermore, whi2Delta and siw14Delta mutants produce large actin clumps in stationary phase similar to those seen in prk1Delta ark1Delta mutants defective in protein kinases that regulate the actin cytoskeleton. Overexpression of SIW14 in a prk1Delta strain resulted in a loss of cortical actin patches and cables and was lethal. Overexpression of SIW14 also rescued the caffeine sensitivity of the slt2 mutant isolated in the screen, but this was not due to alteration of the phosphorylation state of Slt2. These observations suggest that endocytosis and the organization of the actin cytoskeleton are required for the proper response to nutrient limitation. This hypothesis is supported by the observation that rvs161Delta, sla1Delta, sla2Delta, vrp1Delta, ypt51Delta, ypt52Delta, and end3Delta mutations, which disrupt the organization of the actin cytoskeleton and/or reduce endocytosis, have a phenotype similar to that of whi2Delta mutants

Spontaneous Secretion of the Citrullination Enzyme PAD2 and Cell Surface Exposure of PAD4 by Neutrophils

Autoantibodies directed against citrullinated epitopes of proteins are highly diagnostic of rheumatoid arthritis (RA), and elevated levels of protein citrullination can be found in the joints of patients with RA. Calcium-dependent peptidyl-arginine deiminases (PAD) are the enzymes responsible for citrullination. PAD2 and PAD4 are enriched in neutrophils and likely drive citrullination under inflammatory conditions. PADs may be released during NETosis or cell death, but the mechanisms responsible for PAD activity under physiological conditions have not been fully elucidated. To understand how PADs citrullinate extracellular proteins, we investigated the cellular localization and activity of PAD2 and PAD4, and we report that viable neutrophils from healthy donors have active PAD4 exposed on their surface and spontaneously secrete PAD2. Neutrophil activation by some stimulatory agents increased the levels of immunoreactive PAD4 on the cell surface, and some stimuli reduced PAD2 secretion. Our data indicate that live neutrophils have the inherent capacity to express active extracellular PADs. These novel pathways are distinguished from intracellular PAD activation during NETosis and calcium influx-mediated hypercitrullination. Our study implies that extracellular PADs may have a physiological role under non-pathogenic conditions as well as a pathological role in RA

14C11 antibody inhibits major group HRV16 infection <i>in vivo</i>.

<p>Groups of 7 mice were dosed intranasally with 14C11 or isotype control 2 hours prior to intranasal infection with HRV16. (A) Total BAL cells, macrophages, lymphocytes and neutrophils were assessed with differentially cytospin counts day 2 after infection. (B) Levels of proinflammatory cytokines and chemokines IL-1β, IL-6, CXCL1, IFNλ2/3, CXCL11 and CXCL10 in cell-free BAL were determined by MSD or quantitative ELISA 2 days after infection. (C) Proinflammatory cytokines and chemokines IL-1β, IL-6 and CXCL1 in lung homogenate were assessed with MSD 2 days after infection. (D) Groups of 3–5 mice were dosed intranasally with 14C11 or isotype control 2 hours prior to intranasal infection with HRV16 and vRNA in lung tissue was assessed by qPCR at the timepoints indicated. (E) Inflammatory cells in the lungs prior to infection (uninfected) and at 12 hours post-infection (12 p.i.) of huICAM negative (tg−) and huICAM-expressing (tg+) mice without antibody treatment, or for tg+ mice following isotype control antibody (tg+ iso) or 14C11 antibody treatment (tg+ 14C11) assessed by H&E staining Arrows indicate peribronchial cellular inflammation. Data are expressed as mean (± SEM). Significance was assessed by One-way ANOVA test with Bonferroni's Multiple Comparison test as post-test. ***p<0.001 and **p<0.01 vs HRV16 infected transgenic negative mice; ^#p<0.05, ^##p<0.01 and ^###p<0.001 vs HRV16 infected transgenic positive mice. Data are representative of 3 independent experiments each.</p

Effect of systemically dosed 14C11 antibody on HRV16 infection in vivo.

<p>Mice were dosed intravenously with 14C11 24 hours prior to intranasal infection with HRV16 (n = 9 for tg− group; n = 6 for tg+ groups). (A) Total BAL cells, amacrophageslymphocytes and neutrophils were assessed by cytospin 2 days after infection. (B) The chemokines CXCL1, CXCL11 and CXCL10 in BAL were determined by MSD or quantitative ELISA 2 days after infection. Data are expressed as mean (± SEM). Significance was assessed by One-way ANOVA test with Bonferroni's Multiple Comparison test as post-test. **p<0.01 and ***p<0.001 vs HRV16 infected transgenic negative mice; ^#p<0.05, ^##p<0.01 and ^###p<0.001 vs HRV16 infected transgenic positive mice; Data are representative of 3 independent experiments.</p

14C11 binds in domain 1 of human ICAM-1, does not bind to mouse ICAM-1 and does not inhibit human ICAM-1/LFA-1 interaction.

<p>(A)14C11 and (B) 84H10 antibody were tested for binding of antigens (human ICAM-1 (Hu ICAM-1), mouse ICAM-1 (Mu ICAM-1) and a recombinant protein consisting of human ICAM-1 D1 and mouse ICAM-1 D2-5 (Hu1 Mu2-5)). Jurkat E6.1 cells were labelled with Calcein-AM dye. Serial dilutions of the antibodies 14C11, 84H10 and isotype control were added in an ICAM-1-Fc coated plate. (C) PMA and labelled Jurkat E6.1 cells were added into the plate. Binding to LFA-1 was determined by fluorescein detection. Data were analysed by normalising values to no PMA (0% signal) and PMA no antibody (100% signal) controls. Data are a representative of three experiments for (A) and (B) and four to seven experiments for (C). Data are expressed as mean (± SEM).</p

Time course of topically dosed 14C11 antibody-mediated inhibition of major group HRV16 infection.

<p>Mice were dosed intranasally with 14C11 or isotype control 2 hours prior to intranasal infection with HRV16. (A)Total BAL cells, macrophages, lymphocytes and neutrophils were assessed with differentially stained cytospins day 2, 4 and 7 after infection. (B) CXCL1, CXCL10 and CXCL11 in the BAL were determined by MSD or quantitative ELISA. (C) HRV16 specific serum IgG1 and IgG2a on day 7 post-infection. Data are a pool of 2 experiments with n = 4 mice per group each. Data are expressed as mean (± SEM). Significance was assessed by Three-way analysis of variance (A and B) or by One-way ANOVA with Bonferroni's Multiple Comparison test as post-test (C). **p<0.01 and ***p<0.001 vs HRV16 infected transgenic negative mice; ^#p<0.05 and ^###p<0.001 vs HRV16 infected transgenic positive mice.</p

14C11 inhibits major group HRV replication <i>in vitro</i>.

<p>Ohio HeLa cells were pre-incubated with serial dilutions of 14C11 or isotype control and infected with (A) HRV16, (B) HRV14 and (C) minor group HRV25. The antiviral effect of 14C11 was determined by CPE reduction assay and expressed as % of control. (D) IC₅₀s for 14C11 were determined for major HRVs by CPE assay as indicated in. Experiment (A), (B) and (C) were performed in sextuplicates and (D) is a pool of two independent experiments. Data are expressed as mean (± SEM).</p